Plants, in general, contain a myriad of secondary metabolites often synthesized by unique biochemical processes operating only in exotic species. For plant-derived products such as drugs, the 1997 worldwide sales were US$ 10 billion (Rotheim 2002). In many cases the supply of the relevant plant material for these drugs is limited or variable. One approach to developing methods for producing these drugs is to apply the methods of biochemistry, molecular biology and genomics to elucidate the biosynthesis and relevant biosynthetic genes for compounds of value for human health.
With the realization that many of the enzymes involved in natural product biosynthesis represent variations within known classes of enzymes, expressed sequence tag (EST) analysis (combined with heterologous expression) provides a powerful means of identifying their corresponding genes (Cahoon et al. 1999, Gang et al. 2001, Lange et al. 2000 and van de Loo et al. 1995)
One area of interest is bioactive compounds of the tribe Anthemideae in the family Asteraceae (Compositae) (Torrell et al. 1999 and Watson et al. 2000). Anthemideae (Asteraceae, subfamily Asteroideae) is a tribe of 109 genera which includes daisies, chrysanthemums, tarragon, chamomile, yarrow and sagebrushes (Watson et al. 2000). These plants are aromatic in nature resulting from high concentrations of mono- and sesqui-terpenes. Many of the species in this tribe are valued for the health benefits or insecticidal properties.
Of particular interest is artemisinin from Artemisia annua or sweet wormwood. In 1972, Chinese scientists isolated the sesquiterpene lactone containing an endoperoxide group (see FIG. 1) from Artemisia and called it qinghaosu (van Agtmael et al. 1999b). Prior to this sweet wormwood or qinghao had been used in traditional Chinese medicine for centuries. Artemisinin has become very important for the treatment of malaria in Southeast Asia and elsewhere, particularly for multi-drug-resistant falciparum forms of the disease (O'Neill 2005, Rathore et al. 2005, Robert et al. 2002, Wilairatana et al. 2002 and Wu 2002). Since the discovery of artemisinin, a number of semi-synthetic derivatives have been developed for specific applications in malaria treatment.
Malaria remains a serious health problem which affects over 400 million people, especially in Africa and Southeast Asia, causing the deaths in excess of 2 million each year. Increasing resistance of the malaria parasite, Plasmodium falciparum, towards current antimalarial drugs is a cause for concern. The future value of antimalarial drugs based on the artemisinin structure is illustrated by the development by Bayer AG of Artemisone, an artemisinin derivative reported to be 10-30 fold more active than artesunate, for which clinical trials are currently under way. Also, researchers at the Walter Reed Army Institute of Research (USA) are currently developing artelinic acid for intravenous treatment of severe malaria.
Artemisinin is produced in relatively small amounts of 0.01 to 1.0% dry weight, making it and its derivatives relatively expensive (Gupta et al. 2002). Several studies describe the chemical synthesis of the sesquiterpene, but none are an economical alternative for isolation of artemisinin from the plant (Yadav et al. 2003). Therefore a higher concentration in the plant or production in an alternative host is desirable to make artemisinin available as economically as possible, especially for use in the Third World. Knowledge of the biosynthetic pathway and the genes involved should enable engineering of improved production of artemisinin. Alternatively, there is also the possibility of producing intermediates in the pathway to artemisinin which are of commercial value. For example, a compound 15 times more potent in vitro than artemisinin against Plasmodium falciparum has been synthesized from artemisinic alcohol (Jung et al. 2001).
There is evidence that artemisinin is localized to glandular trichomes on the surfaces of certain tissues of the plant (Duke et al. 1994 and Duke et al. 1993). The number and even existence of these trichomes and the amount of artemisinin varies widely among biotypes.
Typically, compounds discovered in plants and found to be useful are produced commercially by i) chemical synthesis, where possible and economical, ii) extraction of cultivated or wild plants, or iii) cell or tissue culture (this is rarely economical). In those cases in which chemical synthesis is not economical, it makes sense to learn as much as possible about the biosynthesis of a natural product, such that it can be produced most efficiently in plants or cell/tissue culture. In the case of artemisinin, chemical synthesis is not commercially feasible. Since the compound is produced in small quantities in Artemisia, the drugs derived from artemisinin are relatively expensive, particularly for the Third World countries in which they are used. While the antimalarial drugs, chloroquine and sulfadoxine-pyrimethamine, cost as little as 20 cents for an adult treatment, artemisinin-derived compounds, by contrast, can be 100 times as expensive. Chloroquine resistance is prevalent and sulfadoxine-pyrimethamine resistance is increasing. The World Health Organization recently added the artemisinin-derived drug, artemether to their Model List of Essential Medicines, which are recommended to be available at all times in adequate amounts and in the appropriate dosage forms, and at a price that individuals and the community can afford. Consequently, it would be useful to be able to supply artemisinin-derived drugs more economically.
There are numerous patents relating to artemisinin and artemisinin derived drugs. These cover drug synthesis and formulation, Artemisia cultivation (Kumar 2002) and tissue culture and artemisinin extraction (Elferaly 1990). Commonly owned U.S. patent application 60/729,210 filed Oct. 24, 2005, the disclosure of which is herein incorporated by reference, and now filed as a PCT patent application, discloses a gene encoding amorpha-4,11-diene hydroxylase, which catalyzes the first committed steps in artemisinin biosynthesis (FIG. 1).
In the past five years a reasonably clear picture of artemisinin biosynthesis has emerged as illustrated in FIG. 1 (Bertea et al. 2005). The identity of amorpha-4,11-diene as a biosynthetic intermediate was established, based on the presence of trace of amorpha-4,11-diene in Artemisia extracts and the cloning and expression of cDNAs representing amorpha-4,11-diene synthase, a terpene cyclase (Bouwmeester et al. 1999 and Wallaart et al. 2001). A cytochrome P450 gene designated cyp71av1 was recently cloned and characterized (Teoh et al. 2006). The cyp71av1 gene encodes a hydroxylase that catalyzes the conversion of amorpha-4,11-diene to artemisinic alcohol. CYP71AV1 expressed in yeast is also capable of oxidizing artemisinic alcohol to artemisinic aldehyde and artemisinic aldehyde to artemisinic acid.